Small linear peptides are useful for investigating various physiological phenomena because they exhibit a wide range of biological activities and can be easily synthesized in almost infinitely variable sequences utilizing conventional techniques in solid phase synthesis and combinatorial chemistry. These qualities also make small linear peptides especially useful for identifying and developing new drugs. For example, large libraries of myriad different small linear peptides can be prepared synthetically and then screened for a particular characteristic in various biological assays. E.g., Scott, J. K. and G. P. Smith, Science 249:386, 1990; Devlin, J. J., et al., Science 24:404, 1990; Furka, A. et al., Int. J. Pept. Protein Res. 37:487, 1991; Lam, K. S., et al., Nature 354:82, 1991. Those peptides within the library that exhibit the particular characteristic can then be isolated as candidates for further study. Microsequencing or other chemical analyses can then be used to characterize selected peptides by, for example, amino acid sequence. Despite these advantages, only a handful of small linear peptides have been developed into widely-used pharmaceutical drugs. One reason for this is that small linear peptides are usually cleared from the body too rapidly to be of therapeutic value.
Ring closure, or cyclization, can reduce the rate at which peptides are degraded in vivo and therefore dramatically improve their pharmocokinetic properties. The majority of cyclic peptides of known therapeutic value have been identified after isolation from natural sources (e.g., calcitonins, oxytocin, and vasopressin). Unfortunately, the pool of naturally-existing cyclic peptides that can be screened for a particular biological activity is inherently limited. And, moreover, the onerous steps required to isolate and purify cyclic peptides from natural sources render such screens costly and impractical. Thus, synthetic methods for producing large numbers of different peptides of infinitely variable amino acid sequences would greatly facilitate identifying particular cyclic peptides as candidates for new drugs.
Various methods for producing cyclic peptides have been described. For example, chemical reaction protocols, such as those described in U.S. Pat. Nos. 4,033,940 and 4,102,877, have been devised to produce circularized peptides. In other techniques, biological and chemical methods are combined to produce cyclic peptides. These latter methods involve first expressing linear precursors of cyclic peptides in cells (e.g., bacteria) to produce linear precursors of cyclic peptides and then adding of an exogenous agent such as a protease or a nucleophilic reagent to chemically convert these linear precursors into cyclic peptides. See, e.g., Camerero, J. A., and Muir, T. W., J. Am. Chem. Society. 121:5597 (1999); Wu, H. et al, Proc. Natl. Acad. Sci. USA, 95:9226 (1998).
Once produced, cyclic peptides can be screened for pharmacological activity. For example, a library containing large numbers of different cyclic peptides can be prepared and then screened for a particular characteristic, such as the ability to bind a specific target ligand. The library is mixed with the target ligand, and those members of the library that bind to the target ligand can be isolated and identified by amino acid sequencing. Similarly, libraries of cyclic peptides can be added to assays for a specific biological activity. Those cyclic peptides which modulate the biological activity can then be isolated and identified by sequencing.
Unfortunately, because the step of identifying the active peptides can be difficult, these screening assays can prove laborious and time-consuming. For instance, screening assays usually mandate a reverse-mapping step because the actual amount of cyclic peptide that binds a target ligand or modulates a biological activity is usually so minute that it cannot be sequenced directly. To avoid this problem, a map indicating the physical location of the various cyclic peptides comprising a library can be made. Aliquots of cyclic peptides from the different locations are then transferred to corresponding locations within the screening assay; and those areas in the assay that exhibit the screened-for activity (e.g., binding or modulation of biological activity) are then mapped back to their corresponding location in the library. The cyclic peptides in that area of the library can then be isolated and sequenced. Difficulties arising from the need for spatial resolution and the limitations imposed by sample handling limit the number of candidate peptides that can be screened in any given period of time.
The number of peptides that can be screened in an assay can be dramatically increased by using cells that express the peptides. For example, bacteria engineered to express a library of linear peptides can be added to a screening assay, and those bacteria that express the screened for characteristic can be picked directly from the assay. The picked bacteria can then be reproduced to large numbers such that the selected linear peptides can made in large quantities to facilitate their identification (e.g., by sequencing) and production. Making and screening small linear peptide libraries in vivo has, however, proven to be troublesome because small linear peptides are rapidly degraded by normal cellular metabolic processes. Cyclization of the peptides can circumvent this problem by rendering the peptides stable within a cell.
Despite this, heretofore, intracellular production of large libraries of cyclic peptides has not been feasible because general, easy-to-perform methods for cyclizing peptides in vivo have not been available. For example, a known method of producing cyclic peptides in vivo utilizes non-ribosomal peptide synthetase (NRPS) complexes (Cane et al, Science 282:63, 1998). Such NRSP complexes are, however, neither facile to work with nor generally useful for the production of more than a single cyclic peptide at a time. Moreover, unlike ribosomal peptide synthesis where the linear sequence of monomers (amino acids) is dictated by the linear sequence of bases in the nucleic acid molecule encoding it, the linear sequence of monomers in a peptide made by the NRPS method is dictated by the subunit organization of the NRPS complex. Changing the sequence of a cyclic peptide made by NRPS entails cloning the subunit(s) which incorporate the desired monomers and introducing the subunit(s) into host cells already harboring all of the other necessary subunits. Making a library using this technique would require introducing combinations (both in composition and order) of NRPS subunits to host cells and devising a method for ensuring that the subunits assemble into the correct supramolecular structures.